G proteins are an important class of regulatory switches in all living systems. They are activated by guanine nucleotide exchange factors (GEFs), which facilitate the exchange of GDP for GTP. This activity makes GEFs attractive targets for modulating disease-relevant G-protein-controlled signalling networks. GEF inhibitors are therefore of interest as tools for elucidating the function of these proteins and for therapeutic intervention; however, only one small molecule GEF inhibitor, brefeldin A (BFA), is currently available. Here we used an aptamer displacement screen to identify SecinH3, a small molecule antagonist of cytohesins. The cytohesins are a class of BFA-resistant small GEFs for ADP-ribosylation factors (ARFs), which regulate cytoskeletal organization, integrin activation or integrin signalling. The application of SecinH3 in human liver cells showed that insulin-receptor-complex-associated cytohesins are required for insulin signalling. SecinH3-treated mice show increased expression of gluconeogenic genes, reduced expression of glycolytic, fatty acid and ketone body metabolism genes in the liver, reduced liver glycogen stores, and a compensatory increase in plasma insulin. Thus, cytohesin inhibition results in hepatic insulin resistance. Because insulin resistance is among the earliest pathological changes in type 2 diabetes, our results show the potential of chemical biology for dissecting the molecular pathogenesis of this disease.

Background:Cytokine secretion has unwanted consequences in malignant and in inflammatory disorders. The deacetylase SIRT6 has pro-inflammatory activity, but the underlying mechanisms and its biological significance remain unclear. The relationship between inflammation and carcinogenesis has been known for many years (1). Chronic inflammation is a risk factor for cancer development. In addition, even in those cancers that do not develop in inflamed tissues, an inflammatory component is usually observed, and it is now known to be an essential part of the malignant microenvironment (2, 3). Inflammation contributes to tumorigenesis and cancer progression by supplying growth factors that sustain cancer cell proliferation and/or survival, proangiogenic factors, extracellular matrix-modifying enzymes that promote invasion and metastasis, and signals that lead to epithelial-mesenchymal transition (2, 4, 5). Moreover, increased circulating levels of pro-inflammatory cytokines are responsible for systemic manifestations of disease, such as cachexia, fever, and sweats (6 -9). Among other forms of cancer, pancreatic ductal adenocarcinoma (PDAC) 2 is well known for its propensity to secrete high levels of pro-inflammatory factors that contribute to its clinical aggressiveness and to its metastatic potential (10). The mechanisms controlling cyto-/chemokine production by inflammatory and cancer cells are only partially understood. A more detailed understanding of the molecular pathways leading to cancer-associated inflammation may lead to new therapeutic strategies with a strong impact on patient quality of life.Previous studies showed that intracellular nicotinamide adenine dinucleotide (NAD ϩ ) levels influence the capacity of inflammatory cells to secrete cytokines, such as tumor necrosis factor ␣ (TNF), interleukin 6 (IL6), IL1␤, interferon ␥ (IFN-␥), * This work was supported in part by the Associazione Italiana per la Ricerca sul Cancro (AIRC, Code 6108) (to A. N.), by the European Seventh Framework Program (Project 256986, PANACREAS) (to A. N.), by Ministero della Salute Grant GR-2008-1135635 (to A. N.)

SIRT6 is an NAD(+)-dependent deacetylase with a role in the transcriptional control of metabolism and aging but also in genome stability and inflammation. Broad therapeutic applications are foreseen for SIRT6 inhibitors, including uses in diabetes, immune-mediated disorders, and cancer. Here we report on the identification of the first selective SIRT6 inhibitors by in silico screening. The most promising leads show micromolar IC50s, have significant selectivity for SIRT6 versus SIRT1 and SIRT2, and are active in cells, as shown by increased acetylation at SIRT6 target lysines on histone 3, reduced TNF-α secretion, GLUT-1 upregulation, and increased glucose uptake. Taken together, these results show the value of these compounds as starting leads for the development of new SIRT6-targeting therapeutic agents.

SUMMARY The fatty-acid ethanolamides (FAEs) are lipid mediators present in all organisms and involved in highly conserved biological functions such as innate immunity, energy balance and stress control. They are produced from membrane N-acylphosphatidylethanolamines (NAPEs) and include agonists for G protein-coupled receptors (e.g. cannabinoid receptors) and nuclear receptors (e.g. PPAR-α). Here we report the crystal structure of human NAPE-hydrolyzing phospholipase D (NAPE-PLD) at 2.65 Å resolution, a membrane enzyme that catalyzes FAE formation in mammals. NAPE-PLD forms homodimers partly separated by an internal ~9 Å-wide channel and uniquely adapted to associate with phospholipids. A hydrophobic cavity provides an entryway for NAPE into the active site, where a binuclear Zn2+ center orchestrates its hydrolysis. Bile acids bind with high affinity to selective pockets in this cavity, enhancing dimer assembly and enabling catalysis. These elements offer multiple targets for the design of small-molecule NAPE-PLD modulators with potential applications in inflammation and metabolic disorders.

Nampt inhibition appears as a new strategy to dampen CXCL2-induced neutrophil recruitment and thereby reduce neutrophil-mediated tissue injury in mice.

The ceramides are a family of bioactive lipid-derived messengers involved in the control of cellular senescence, inflammation, and apoptosis. Ceramide hydrolysis by acid ceramidase (AC) stops the biological activity of these substances and influences survival and function of normal and neoplastic cells. Because of its central role in the ceramide metabolism, AC may offer a novel molecular target in disorders with dysfunctional ceramide-mediated signaling. Here, a class of benzoxazolone carboxamides is identified as the first potent and systemically active inhibitors of AC. Prototype members of this class inhibit AC with low nanomolar potency by covalent binding to the catalytic cysteine. Their metabolic stability and high in vivo efficacy suggest that these compounds may be used as probes to investigate the roles of ceramide in health and disease, and that this scaffold may represent a promising starting point for the development of novel therapeutic agents.

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The fatty acid amide hydrolase (FAAH) regulates the endocannabinoid system cleaving primarily the lipid messenger anandamide. FAAH has been well characterized over the years and, importantly, it represents a promising drug target to treat several diseases, including inflammatory-related diseases and cancer. But its enzymatic mechanism for lipid selection to specifically hydrolyze anandamide, rather than similar bioactive lipids, remains elusive. Here, we clarify this mechanism in FAAH, examining the role of the dynamic paddle, which is formed by the gating residues Phe432 and Trp531 at the boundary between two cavities that form the FAAH catalytic site (the “membrane-access” and the “acyl chain-binding” pockets). We integrate microsecond-long MD simulations of wild type and double mutant model systems (Phe432Ala and Trp531Ala) of FAAH, embedded in a realistic membrane/water environment, with mutagenesis and kinetic experiments. We comparatively analyze three fatty acid substrates with different hydrolysis rates (anandamide > oleamide > palmitoylethanolamide). Our findings identify FAAH’s mechanism to selectively accommodate anandamide into a multi-pocket binding site, and to properly orient the substrate in pre-reactive conformations for efficient hydrolysis that is interceded by the dynamic paddle. Our findings therefore endorse a structural framework for a lipid selection mechanism mediated by structural flexibility and gating residues between multiple binding cavities, as found in FAAH. Based on the available structural data, this exquisite catalytic strategy for substrate specificity seems to be shared by other lipid-degrading enzymes with similar enzymatic architecture. The mechanistic insights for lipid selection might assist de-novo enzyme design or drug discovery efforts.